key: cord-315702-pn8247j2
authors: Sahakijpijarn, Sawittree; Moon, Chaeho; Koleng, John J.; Christensen, Dale J.; Williams, Robert O.
title: Development of Remdesivir as a Dry Powder for Inhalation by Thin Film Freezing
date: 2020-09-22
journal: bioRxiv
DOI: 10.1101/2020.07.26.222109
sha: 
doc_id: 315702
cord_uid: pn8247j2

Remdesivir exhibits in vitro activity against SARS-CoV-2 and was granted approval for Emergency Use. To maximize delivery to the lungs, we formulated remdesivir as a dry powder for inhalation using thin film freezing (TFF). TFF produces brittle matrix nanostructured aggregates that are sheared into respirable low-density microparticles upon aerosolization from a passive dry powder inhaler. In vitro aerodynamic testing demonstrated that drug loading and excipient type affected the aerosol performance of remdesivir. Remdesivir combined with optimal excipients exhibited desirable aerosol performance (up to 93.0% FPF; 0.82μm MMAD). Remdesivir was amorphous after the TFF process, which benefitted drug dissolution in simulated lung fluid. TFF remdesivir formulations are stable after one-month storage at 25 °C/60%RH. In vivo pharmacokinetic evaluation showed that TFF-remdesivir-leucine was poorly absorbed into systemic circulation while TFF-remdesivir-Captisol® demonstrated increased systemic uptake compared to leucine. Remdesivir was hydrolyzed to the nucleoside analog GS-441524 in lung, and levels of GS-441524 were greater in lung with the leucine formulation compared to Captisol®. In conclusion, TFF technology produces high potency remdesivir dry powder formulations for inhalation suitable to treat patients with COVID-19 on an outpatient basis and earlier in the disease course where effective antiviral therapy can reduce related morbidity and mortality.

The coronavirus disease 2019 (COVID-19) is an ongoing worldwide pandemic. As of

September 2020, laboratory-confirmed cases have been reported in 213 countries and territories with 29 more than 30 million reported cases and close to 1 million reported deaths [1] . Although this disease 4 of 39 antiviral activity in the lungs, and limit the potential for systemic side effects [18] . In addition, the cost 89 of the drug can be reduced, and the supplies of the drug can be maximized, thus treating more 90 patients due to less dose required by inhalation as compared to injectable forms. The treatment cost 91 can also be decreased when administered by inhalation, since patients may not need to visit hospitals 92 as is required to administer the IV injectable dose. Therefore, more affordable and early stage 93 treatment can be provided to patients with inhaled remdesivir.

Nebulization of the current IV formulation in a diluted form is a potential method of pulmonary 95 administration; however, the drug is prone to degrade by hydrolysis in aqueous solution to form the 96 nucleoside monophosphate, which has difficulty penetrating cell membranes, thereby minimizing the 97 antiviral activity in the lung cells [19] . Another concern is the use of SBECD as an excipient in 

Although several techniques have been used to prepare inhalable powders, including 116 mechanical milling and spray drying, the advantages of thin film freezing (TFF) over other techniques 5 of 39 rely on the ability to produce aerosolizable particles composed of brittle matrix, nanostructured 118 aggregates. These are high surface area powders that are ideally suited for dry powder inhalation.

TFF employs ultra-rapid freezing (on the order of 100-1,000 K/sec) such that precipitation (either as 120 a crystalline nanoaggregate or amorphous solid dispersion) and particle growth of the dissolved 121 solute can be prevented [25] . Subsequently, nanostructured aggregates are formed as a low-density 122 brittle matrix powder [26] , which is efficiently sheared into respirable low-density microparticles by a 

The EF was calculated as the total amount of remdesivir emitted from the device as a percentage of 

The pan with a T-zero hermetic lid were crimped, and a hole was drilled in the lid before placing the 

The dispersion was adjusted to 10 mL by adding 1M HBSS). The dispersion was frozen at -20 C for 

Intratracheal administration was carried out using the dry powder insufflator (DP-4M model,

Penn-Century Inc., Philadelphia, PA) connected to the air pump (AP-1 model, Penn-Century Inc.,

Philadelphia, PA The aerodynamic particle size distribution of TFF remdesivir formulations was evaluated 374 using a Plastiape® RS00 high resistance DPI and NGI apparatus. Figure 4 and 

1H-NMR was performed to identify interactions between remdesivir and excipients. Figure 5B 417 demonstrates an expansion of 1H-NMR spectra for selected TFF remdesivir powder formulations and 418 remdesivir unprocessed powder. While the peak at 6.03 ppm is sharp and does not show any 419 differences from the presented samples, the peaks at 5.37 and 6.33 ppm of F9 exhibited broader 420 peak. Also, these peaks were slightly shifted to downfield. Figure 5C shows 

The RS00 high resistance monodose DPI is a capsule-based DPI device that is available for 515 commercial product development, and it functions to disperse the powder based on impaction force.

A previous study confirmed that this impact-based DPI can disperse low-density brittle matrix powders 517 made by TFF process into respirable particles better than a shear-based DPI (e.g., Handihaler®) [36] .

Another study also evaluated the performance of different models of the monodose DPI (RS01 and 519 RS00) on the aerosol performance of brittle matrix powders containing voriconazole nanoaggregates 520 prepared by TFF [37] . It was shown that the RS00 device exhibited better powder shearing and 521 deaggregation through smaller holes of the capsule created by the piercing system of the RS00 522 device [37]. Therefore, the RS00 high resistance Plastiape® DPI was selected for this study.

We found that excipient type and drug loading affect the aerosol performance of TFF 524 remdesivir powder compositions. Overall, the aerosol performance of TFF remdesivir powders 525 increased as the drug loading was increased, a highly desirable feature. This trend is obvious for the 526 Captisol®-, lactose-, and mannitol-based formulations when the drug loading was increased from 20% 527 to 50%. Furthermore, high potency TFF remdesivir powder without excipients (F14 and F15) also 528 exhibited high FPF and small MMAD, which indicates remdesivir itself has a good dispersing ability 529 without the need of a dispersing excipient when prepared using the TFF process. This shows that the 530 TFF technology can be used to minimize the need of excipient(s) in the formulation, thus maximizing 531 the amount of remdesivir being delivered to the lungs by dry powder inhalation.

The aerosol performance of the leucine-based formulations did not significantly change when 533 the drug loading was increased from 20% to 80%, and these formulations exhibited superior aerosol 534 performance compared to the other excipient-based formulations studied in this paper. This is likely 

One potential concern related to the amorphous drug is physical instability due to its high 561 energy state. According to criteria described by Wyttenbach 

In addition to the physical stability, remdesivir, as a prodrug, is prone to degrade by hydrolysis 579 in aqueous solution. Since an organic/water co-solvent system is required to dissolve the drug and 580 excipients in the TFF process, chemical stability is another concern during preparation. NMR spectra 581 demonstrated that remdesivir did not chemically degrade as a result of the TFF process. Even though 582 remdesivir was exposed to binary co-solvent systems consisting of water during the process, the 583 entire TFF process used to produce remdesivir dry powder inhalation formulations did not induce 584 chemical degradation of the parent prodrug. Furthermore, remdesivir was chemically stable by HPLC

(data not shown) and NMR after one-month storage at 25C/60%RH. 

Since remdesivir is a poorly water-soluble drug, its dissolution may be a critical factor of drug 589 release in the lung fluid, especially in high drug load formulations. Undissolved particles can be 590 cleared by mucociliary clearance or macrophage uptake, causing lower drug concentration, lung 591 irritation, and inflammatory response [46] . Therefore, a dissolution test was evaluated in this study.

The dissolution profile demonstrated that physical form of drug appears to have a significant effect Generally, even though leucine is a hydrophobic amino acid, it is a water-soluble excipient that has 627 higher solubility than poorly water-soluble drugs like remdesivir. Therefore, the interactions between 628 drug and leucine at the molecular level can increase the dissolution rate of poorly water-soluble drug 629 [53, 54] , like that observed in our study .

Significantly higher plasma levels of GS-441524 than remdesivir were observed in our rat 631 pharmacokinetic study. The half-life of remdesivir is reportedly much shorter than that of GS-441524.

While half-life of GS-441524 is approximately 24.5 hours, for remdesivir it is only about 1 hour in 633 humans following multiple once-daily IV administrations [3] . In rats, the half-life of remdesivir in 634 plasma is reported to be less than 0.9 minute, which is much shorter than that in humans, due to 

We have demonstrated that low density, highly porous brittle matrix particles of remdesivir 

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